CN112516300A

CN112516300A - Anti-tumor vaccine molecule, and preparation method and application thereof

Info

Publication number: CN112516300A
Application number: CN202011415734.8A
Authority: CN
Inventors: 郭军; 杨光富; 杜晶晶; 王昌伟
Original assignee: Central China Normal University
Current assignee: Central China Normal University
Priority date: 2019-12-04
Filing date: 2020-12-04
Publication date: 2021-03-19
Also published as: WO2021110120A1

Abstract

The invention relates to the field of anti-tumor vaccines, and discloses an anti-tumor vaccine molecule, and a preparation method and application thereof. The anti-tumor vaccine molecule has a structure shown in a formula (I), wherein A is an adjuvant, B is an antigen, m A are respectively and covalently connected with protein through covalent connecting arms, and n B are respectively and covalently connected with protein through covalent connecting arms. The anti-tumor vaccine is a novel anti-tumor molecule, has good immunity, can generate IgG antibody with higher titer and stronger cellular immunity, has good thermal stability, and is easy to store and transport.A_m-protein-B_nFormula (I).

Description

Anti-tumor vaccine molecule, and preparation method and application thereof

Technical Field

The invention relates to the field of anti-tumor vaccines, in particular to an anti-tumor vaccine molecule and a preparation method and application thereof.

Background

Extensive studies in cell biology and biochemistry indicate that tumor associated antigens (TACAs, e.g., MUC1 glycoprotein) are ideal targets for immunotherapy and are being vigorously pursued in the development of immunotherapeutic anti-cancer strategies.

To date, immunotherapy has proven to be one of the most promising cancer treatments, offering many possibilities.

However, since the commonly used tumor-associated antigens are self-antigens, they have poor antigenicity and poor immunoregulatory tolerance, resulting in failure to elicit an effective immune response. Therefore, it is an urgent problem to improve the immunogenicity of tumor-associated antigens.

Numerous studies have been undertaken to obtain more immunogenic tumor-associated antigens, as well as linkers that link the antigen to different immunostimulatory components: completely synthesized two-component anticancer vaccine containing built-in adjuvant, three-component anticancer vaccine containing Th cell epitope and built-in adjuvant or multi-component anticancer vaccine containing Th and Tc cell epitope and built-in adjuvant, wherein the commonly used adjuvant comprises Toll-like receptor 2 lipopeptide ligand (Pam)₃CSK₄) Monophosphoryl lipid a (mpla), CpG, NKT cell agonists (α GalCer), and the like. Animal experiment results show that the immune response of mice to tumor-associated antigens may be gradually enhanced with the increase of the amount of vaccine components, but the synthesis difficulty is further increased.

Further, there have also been studies disclosing vaccines equipped with optimized non-covalently linked MUC1 or nicotine antigen and α GalCer, significantly improving the immunogenicity of the antigen.

In addition, semi-synthetic anti-cancer vaccines are typically composed of tumor-associated antigens conjugated to different carrier proteins, including Bovine Serum Albumin (BSA), CRM197 (diphtheria toxin non-toxic mutant), Tetanus Toxoid (TTOX), and Keyhole Limpet Hemocyanin (KLH), and then mixed with adjuvants. Since the carrier protein has a plurality of Tc and Th epitopes, the antigen presentation can be enhanced, and the immune response of the vaccine can be easily improved.

However, to develop more effective anti-tumor immunotherapy strategies, current challenges still exist.

Also, Toll-like receptors (TLRs) are intracellular pattern recognition receptors that recognize highly conserved components of multiple pathogens, inducing innate and adaptive immune responses in the host. Second, it has the potential to modulate the activation of antigen presenting cells, enhancing the secretion of co-stimulatory molecules and many cytokines.

Disclosure of Invention

The invention aims to overcome the problem of poor immunogenicity of tumor-associated antigens in the prior art.

In order to achieve the above object, a first aspect of the present invention provides an anti-tumor vaccine molecule having a structure represented by formula (I), wherein a is an adjuvant, B is an antigen, and m of the a are covalently linked to the protein through covalent linking arms, respectively, and n of the B are covalently linked to the protein through covalent linking arms, respectively, wherein the number of amino acid molecules in the protein is 100 or more; m in the formula (I) is an integer which is more than or equal to 1, and n is an integer which is more than or equal to 1;

A_m-protein-B_nFormula (I).

In a second aspect, the present invention provides a method for preparing an anti-tumor vaccine molecule having a structure represented by formula (I) as defined in the first aspect, the method comprising:

first coupling an antigen to a protein and second coupling the resulting first intermediate to an adjuvant; or

The adjuvant is third coupled to the protein and the resulting second intermediate is fourth coupled to the antigen.

The third aspect of the present invention provides the use of the anti-tumor vaccine molecule having the structure represented by formula (I) in the first aspect in an anti-tumor vaccine.

Polypeptides and proteins used in vaccines differ significantly in structure, preparation and properties: (1) the number of the constituent amino acids of the polypeptide is small (<100, generally used polypeptide is less than 50 amino acids), and the molecular weight is small; the protein has more amino acids (>100), large molecular weight and large volume; (2) the polypeptide is generally synthesized chemically, can be modified at fewer sites, is easy to modify in the chemical synthesis process or after synthesis, and can be purified by HPLC; proteins are generally biosynthesized, have multiple modifiable sites, are mainly modified in a water phase after synthesis and cannot be purified by HPLC; (3) the polypeptide has small volume, is easy to diffuse and is not easy to enrich in lymph tissue, the number of Th and Tc epitopes contained is generally less than 3, and the immunogenicity is weaker; the protein has large volume, is not easy to diffuse, is easy to enrich in lymphatic tissues, contains more Th and Tc epitopes and has stronger immunogenicity.

The design of the strategy of the protein conjugate embedded with the adjuvant is an effective strategy for designing high-efficiency immunotherapy anti-cancer vaccines. Compared with the traditional vaccine strategy, the anti-tumor vaccine is a novel anti-tumor molecule, has good immunity, can generate IgG antibody with higher titer and stronger cellular immunity, has good thermal stability, and is easy to store and transport.

Drawings

FIG. 1 is a preferred embodiment anti-tumor vaccine molecule according to the present invention, wherein FIG. 1A shows the anti-tumor vaccine molecule according to the preferred embodiment of the present invention, in FIG. 1A, the immuno-agonist is an adjuvant, and LK1 and LK2 both represent covalent linking arms; fig. 1B shows an anti-tumor vaccine molecule according to another more preferred embodiment of the present invention, in fig. 1B, the TLR agonist is an adjuvant, and both LK1 and LK2 represent covalent linking arms.

FIG. 2 shows the cytokine IFN-. gamma.and IL-6 assays for sera taken 2h after the first immunization described in example 9.

Figure 3 shows the results of the IgG antibody titer test against MUC1 for the triones described in example 10.

Figure 4 shows a comparison of the IgG antibody titer test results for primary, secondary and tertiary immunizations against MUC1 as described in example 10.

Figure 5 shows a comparison of the results of IgM antibody titer tests against MUC1 for the primary, secondary and tertiary immunizations described in example 10.

Figure 6 shows a comparison of the results of the IgG subtype antibody titer test against MUC1 for the triones described in example 10.

FIG. 7 shows a comparison of the results of the IgG antibody titer test for the triatomines against BSA described in example 11.

FIG. 8 shows the viability of MCF-7 cells determined by the MTT method described in example 12.

FIG. 9 shows the flow cytometry analysis of vaccine-induced antisera binding to MCF-7 cells as described in example 13.

FIG. 10 shows the lysis rates of MCF-7 cells described in example 15.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

As described above, the first aspect of the present invention provides an anti-tumor vaccine molecule having a structure represented by formula (I), wherein a is an adjuvant, B is an antigen, and m of the as are covalently linked to the protein through at least one covalent linking arm, respectively, and n of the as are covalently linked to the protein through at least one covalent linking arm, respectively, the number of amino acid molecules in the protein is 100 or more; m in the formula (I) is an integer which is more than or equal to 1, and n is an integer which is more than or equal to 1;

A_m-protein-B_nFormula (I).

According to a first preferred embodiment, m in said formula (I) is 1, n being an integer greater than or equal to 1; for example, m is 1, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and the like.

According to a second preferred embodiment, m in said formula (I) is 1, n being an integer greater than or equal to 2; for example, m is 1 and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

According to a third preferred embodiment, m in formula (I) is an integer greater than or equal to 2, n is an integer greater than or equal to 1; for example, m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc., and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.

According to a fourth preferred embodiment, m in formula (I) is an integer of 2 or more, and n is an integer of 2 or more; for example, m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc., and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.

In the present invention, the adjuvant and the protein can be covalently linked to each other through any covalently linkable site, and the antigen and the protein can also be covalently linked to each other through any covalently linkable site.

Preferably, the antigen comprises at least one of a tumor-associated antigen, a tumor-specific antigen, a pathogen antigen, a biotoxin, and a biomolecule antigen.

The tumor-associated antigen of the present invention refers to an antigenic molecule present on a tumor cell or a normal cell, and includes, for example: embryonic proteins, glycoprotein antigens, squamous cell antigens, and the like, are commonly used for clinical diagnosis of tumors. Tumor-associated antigens are not specific to tumor cells, are synthesized in minute quantities by normal cells, and are highly expressed when tumor cells proliferate, and are therefore referred to as "associated antigens".

The tumor specific antigen of the present invention refers to a novel antigen that is expressed only on the surface of a certain tumor cell and is not present on a normal cell, and is also called a unique tumor antigen.

Preferably, the tumor-associated antigen is selected from at least one of a tumor-associated polypeptide antigen, a tumor-associated glycopeptide antigen and a tumor-associated glycopeptide antigen.

Preferably, the antigen contains at least one polypeptide or glycopeptide selected from MUC1, MUC16, NY-ESO-1, MAGE-A1/3/4, WT1, STAT3, HER2 and GP 100.

The MUC1 of the present invention is a glycosylation modified, high molecular weight (Mr > 200X 103) mucin 1, which is a transmembrane molecule with a transmembrane sequence such as a rod inserted into the cell membrane and a tail consisting of 69 amino acid residues extending into the cytoplasm.

The MUC16, also named CA125, is a high molecular weight glycoprotein expressed on the surfaces of various epithelial cells, and mainly plays a role in protecting and repairing the epithelium.

The NY-ESO-1 represents the esophageal squamous cell carcinoma of New York 1(NY-ESO-1), is a cancer-testis antigen (CTA) and is re-expressed in a plurality of tumors.

The MAGE-A1/3/4 of the present invention is indicated for detecting tumor-testis antigen (CTA) melanin antigen A (MAGE-A).

WT1 of the present invention represents a Wilms tumor protein, which is expressed by the human WT1 gene.

STAT3 of the present invention represents signaling and activating transcription factor 3.

The HER2 of the invention represents human epidermal growth factor receptor-2 (HER 2).

The GP100 of the present invention represents a melanin-associated antigen.

Preferably, the antigen comprises MUC1, more preferably the MUC1 antigen is selected from at least one of the following structures:

wherein, in the structure of the antigen containing MUC1, each R¹、R²、R³、R⁴And R⁵All attached are amino acid residue modifying groups, and each R¹、R²、R³、R⁴And R⁵Each independently selected from hydrogen and the sugar structures shown below:

preferably, the tumor-associated carbohydrate antigen is selected from at least one of the following structures:

in the anti-tumor vaccine molecule according to the present invention, the protein is preferably at least one selected from the group consisting of Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA), Keyhole Limpet Hemocyanin (KLH), Tetanus Toxoid (TT), Diphtheria Toxoid (DT), haemophilus influenzae D protein, meningococcal outer membrane protein complex group B (OMP), pertussis toxoid, typhoid bacillus flagellae, Pneumolysin (PLY), and a nontoxic diphtheria toxin mutant (CRM 197).

Preferably, in the anti-tumor vaccine molecule of the present invention, the adjuvant is a pattern recognition receptor agonist.

Preferably, the pattern recognition receptor agonist is selected from at least one of a Toll-like receptor agonist and an NKT agonist; more preferably, the Toll-like receptor agonist is selected from at least one of a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR3 agonist, a TLR2 agonist and a TLR4 agonist.

Particularly preferred, representative structures of TLR7 agonists are shown below:

wherein the content of the first and second substances,

in the above representative structures of the TLR7 agonists, R₁Any one selected from the following structures:

in the above representative structures of the TLR7 agonists, R₂Any one selected from the following structures:

in other preferred cases, representative structures of TLR7 agonists are shown below:

particularly preferred, representative structures of TLR8 agonists are as follows:

particularly preferred TLR9 agonists are CpG-ODN, representative structures are as follows:

wherein 5- (P ═ S) TCCATGACGTTCCTGACGT represents a nucleic acid sequence.

Particularly preferred TLR3 agonists are poly (I: C) and poly-ICLC, representative structures being as follows:

poly (I: C) is polymyosamic acid, polymyosamic acid-polycystic acid, an analog of double-stranded RNA, one strand being Poly (I) and the other strand being Poly (C).

Poly-ICLC is a synthetic double-stranded polyriboinosine-polyribocytidylic acid Poly (I: C) stabilized with polylysine and carboxymethylcellulose (LC).

Particularly preferred, representative structures of TLR2 agonists are as follows:

particularly preferred, representative structures of TLR4 agonists are as follows:

R₃represents a hydrogen atom or a phosphoric acid group;

R₄、R₅、R₆、R₇each independently represents a fatty acyl group having 1 to 20 carbon atoms;

R₈represents a hydrogen atom or a phosphoric acid group;

R₉represents hydroxy, amino or carboxyl;

a. b, c and d represent alkyl with 1-20 carbon numbers.

R⁸represents a hydrogen atom or a phosphoric acid group;

R⁹represents hydroxyl, amino, carboxyl and phosphate;

R¹⁰represents a hydrogen atom or a methyl group, including the R/S configuration;

R¹¹and R¹²Each independently represents a hydroxyl group, an amino group, a carboxyl group;

R¹³、R¹⁴and R¹⁵Each independently represents a fatty acyl group having 1 to 20 carbon atoms;

x represents an oxygen or sulphur or selenium atom or an amino group;

z represents oxygen or amino;

e. f and g represent alkyl of 1-20 carbon number;

h. i, j and k each independently represent an integer of 0 to 6.

Particularly preferred, NKT cell agonists have the following representative structures:

preferably, in the anti-tumor vaccine molecule of the present invention, the structure of each covalent linking arm is independently selected from the following structures:

-CO-、-O-CO-、-NH-CO-、-NH(C＝NH)-、-SO₂-、-O-SO₂-、-NH-、-NH-CO-CH₂-、-CH₂-、-C₂H₄-、-C₃H₆-、-C₄H₈-、-C₅H₁₀-、-C₆H₁₂-、-C₇H₁₄-、-C₈H₁₆-、-C₉H₁₈-、-C₁₀H₂₀-、-CH(CH₃)-、-C[(CH₃)₂]-、-CH₂-CH(CH₃)-、-CH(CH₃)-CH₂-、-CH(CH₃)-C₂H₄-、-CH₂-CH(CH₃)-CH₂-、-C₂H₄-CH(CH₃)-、-CH₂-C[(CH₃)₂]-、-C[(CH₃)₂]-CH₂-、-CH(CH₃)-CH(CH₃)-、-C[(C₂H₅)(CH₃)]-、-CH(C₃H₇)-、-(CH₂-CH₂-O)_p-CH₂-CH₂-、-CO-CH₂-、-CO-C₂H₄-、-CO-C₃H₆-、-CO-C₄H₈-、-CO-C₅H₁₀-、-CO-C₆H₁₂-、-CO-C₇H₁₄-、-CO-C₈H₁₆-、-CO-C₉H₁₈-、-CO-C₁₀H₂₀-、-CO-CH(CH₃)-、-CO-C[(CH₃)₂]-、-CO-CH₂-CH(CH₃)-、-CO-CH(CH₃)-CH₂-、-CO-CH(CH₃)-C₂H₄-、-CO-CH₂-CH(CH₃)-CH₂-、-CO-C₂H₄-CH(CH₃)-、-CO-CH₂-C[(CH₃)₂]-、-CO-C[(CH₃)₂]-CH₂-、-CO-CH(CH₃)-CH(CH₃)-、-CO-C[(C₂H₅)(CH₃)]-、-CO-CH(C₃H₇) -or-CO- (CH)₂-CH₂-O)_p-CH₂-CH₂-；

Wherein, in the structure of the covalent linking arm,

each x is independently selected from an integer from 1 to 60;

each Y is independently selected from at least one of-NH-, -O-, -S-and-S-S-;

each p is independently selected from an integer from 1 to 60.

The method for preparing the anti-tumor vaccine molecule is not particularly limited, and those skilled in the art can determine a suitable method to prepare the anti-tumor vaccine molecule by combining the structural characteristics of the anti-tumor vaccine molecule in the art and the conventional synthetic method in the art, and the example section of the present invention exemplifies a specific preparation method of a part of the anti-tumor vaccine molecule, and those skilled in the art can also determine a specific preparation method of all the anti-tumor vaccine molecules in the present invention by combining the preparation methods exemplarily recited in the present invention, however, those skilled in the art should not be construed as limiting the present invention.

Similarly, the adjuvants, antigens and proteins of the anti-tumor vaccine molecules of the present invention can be obtained either synthetically by using existing methods or commercially available, and the present invention is not limited thereto.

Preferably, as mentioned above, the second aspect of the present invention provides a method of preparing the anti-tumour vaccine molecule of the first aspect of the present invention, the method comprising:

It should be noted that the foregoing "first", "second", "third", "fourth", etc. are used for distinguishing only, which means that the above definitions are not the same process, but do not indicate a sequential order, and those skilled in the art should not be construed as limiting the present invention.

As mentioned above, the method for preparing the anti-tumor vaccine molecule of the present invention has at least two methods. The first one is: the antigen is first coupled to the protein and the resulting first intermediate is second coupled to the adjuvant. The second method is as follows: the adjuvant is third coupled to the protein and the resulting second intermediate is fourth coupled to the antigen.

The present invention is not particularly limited with respect to specific conditions for each coupling, and those skilled in the art will be able to determine appropriate conditions in view of the context of the present invention and the routine practice in the art.

As mentioned above, a third aspect of the present invention provides the use of an anti-tumour vaccine molecule as described in the previous first aspect of the invention in an anti-tumour vaccine.

Several preferred embodiments of the present invention are provided below.

In a preferred embodiment of the present invention, the present invention provides an anti-tumor vaccine molecule with the structure shown in fig. 1A, wherein the antigen molecule and the adjuvant molecule are covalently linked to the same protein (i.e. carrier protein) to obtain the three-in-one protein conjugate with embedded adjuvant, which can generate immune response of high-titer IgG antibodies when used as an anti-tumor vaccine molecule. As shown in FIG. 1A, in the structural formula of the anti-tumor vaccine molecule, LK1 and LK2 both represent covalent linking arms. In this preferred embodiment, the adjuvant, antigen, protein, covalent linking arm are as described above.

In particular, the inventors of the present invention found that when a TLR7 agonist is used as an adjuvant, the immunostimulatory activity can be significantly better enhanced and side effects reduced. Thus, in another more preferred embodiment of the present invention, TLR7 agonist is used as adjuvant and BSA is used as protein, which is covalently bound to the tumor associated glycopeptide antigen MUC1 three components to form an adjuvant-protein-antigen vaccine molecule, the specific structure of which is shown in fig. 1B, wherein both LK1 and LK2 represent covalent linking arms. In this preferred embodiment, the antigen, protein, covalent linker arm are as described above. The anti-tumor vaccine molecule not only produces significant IgG antibodies, but also induces relatively high levels of IgG2a, resulting in a bias in antibody type towards Th1 type cellular immunity.

The invention provides a three-in-one protein combined vaccine strategy with a novel structure, namely, an adjuvant is covalently combined on a carrier protein combined with an antigen, and the scheme is applied to the field of anti-cancer (namely anti-tumor) vaccines for the first time.

In a preferred embodiment, the present invention also has the following specific advantages:

(1) due to the clustered arrangement of the conjugated TLR7 agonist on the carrier protein-antigen conjugated conjugate, the pharmacokinetic properties will be greatly altered, transport of the conjugate to lymph nodes is facilitated, immunostimulatory activity is enhanced and side effects are reduced.

(2) Activating Antigen Presenting Cells (APCs) and activating T cells.

(3) High affinity IgG antibodies to the antigen and higher IgG2a antibodies were generated.

(4) The antibodies produced recognize cancer cells and can initiate lysis of the recognized cancer cells by activating Complement Dependent Cytotoxicity (CDC) of, for example, rabbit serum.

(5) Triggering cytotoxic T lymphocyte killing effect (CTL), mediating stronger T cell immunity.

The three-in-one protein conjugate (namely the anti-tumor vaccine molecule) provided by the invention is an effective strategy for designing an efficient immunotherapy anti-cancer vaccine.

The present invention will be described in detail below by way of examples. In the following examples, the test methods used, unless otherwise specified, were conventional. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: preparation of TLR7 agonist

Synthesis of Compound 2: 2-chloroadenine (5.4g,31.8mmol) and sodium (5.0g,217mmol) were mixed in ethylene glycol monomethyl ether (235mL,3.1mol), and the mixture was stirred at 140 ℃ under reflux for 10 h. Then, 25mL of water was added, 1mol/L hydrochloric acid was added to pH 7, and the mixture was concentrated, washed with water and filtered with suction to obtain compound 2, which was carried out in the next step without purification.

Synthesis of compound 3: compound 2(0.7g,3.34mmol), potassium carbonate (3.2g,23.44mmol), methyl 4-bromomethylbenzoate (1.5g,6.68mmol) were added to 10mL dry N, N-Dimethylformamide (DMF) and stirred at 60 ℃ under reflux for 8 h. Adding 5 wt% citric acid water solution until no bubble is generated, extracting with chloroform, washing with brine, and MgSO₄Drying, filtering and concentratingAnd (4) purifying the reaction mixture by column chromatography to obtain a white solid 3.¹H NMR(400MHz,DMSO-d₆) δ (ppm)8.10(s,1H),7.95(d, J ═ 8.0Hz,2H),7.43(s,1H),7.42(d, J ═ 8.1Hz,2H,2 × benzene ring), 7.30(s,2H,2 × benzene ring), 5.38(s,2H, NH₂),4.32(t,J＝4.7Hz,2H,2H,CH₂-O),3.85(s,3H,CH₃-O),3.61(t,J＝4.7Hz,2H,2H,CH₂-O),3.28(s,3H,CH₃-O).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)166.40(C＝O),161.82(C₂),157.23(C₆),151.66(C₄),142.93(C₈) 140.03,130.02,129.38,128.23 (benzene), 115.59 (C)₅),70.74(O-CH₂-),65.82(-CH₂-O),58.55(-OCH₃),52.64(-OCH₃),46.18(CH₂-C₆H₄COOH) HRMS calcd C₁₇H₂₀N₅O₄ ⁺[M+H]⁺358.1510, found 358.1513.

Synthesis of compound 4: compound 3(0.8g,2.2mmol) was added to chloroform (10mL), and elemental bromine (227. mu.L, 4.4mmol) was added and stirred at room temperature for 8 h. Then adding saturated Na₂S₂O₃Removing excess bromine, extracting with chloroform, washing the organic phase with brine, MgSO₄Drying, filtering, concentrating, and purifying the reaction mixture by column chromatography to obtain a white solid 4.¹H NMR(400MHz,DMSO-d₆) Delta (ppm):7.96(d, J ═ 8.1Hz,2H,2 Xbenzene ring), 7.50(s,2H, NH)₂) 7.36(d, J ═ 8.1Hz,2H,2 × phenyl ring), 5.36(s,2H, CH)₂-C₆H₄CO₂CH₃),4.33(t,J＝4.7Hz,2H,CH₂-O),3.85(s,3H,CH₃-O),3.61(t,J＝4.7Hz,2H,CH₂-O),3.29(s,3H,CH₃-O).¹³C NMR(101MHz,DMSO-d₆)δ(ppm):166.34(C＝O),161.86(C₂),156.20(C₆),152.92(C₄) 141.78,130.13,129.51,127.82 (benzene), 124.29 (C)₈),115.87(C₅),70.68(O-CH₂-),66.02(-CH₂-O),58.56(-OCH₃),52.66(-OCH₃),46.59(CH₂-C₆H₄COOH) HRMS calculated valueC₁₇H₁₉BrN₅O₄ ⁺[M+H]⁺436.0615, found 436.0619.

Synthetic compound 5 (defined as TLR7 a): compound 4(0.5g) was added to 20ml of 6M NaOH: MeOH 4:1(v/v) and stirred at 100 ℃ under reflux for 4 h. Then, 1mol/L hydrochloric acid was added to adjust the pH to 7, followed by concentration, water washing, dissolution of the solid with methanol, and purification of the reaction mixture by column chromatography to obtain compound 5.¹H NMR(400MHz,DMSO-d₆)δ(ppm):10.09(s,1H,CO₂H) 7.90(d, J ═ 7.9Hz,2H,2 × phenyl ring), 7.37(d, J ═ 7.9Hz,2H,2 × phenyl ring), 6.53(s,2H, NH₂),4.92(s,2H,CH₂-C₆H₄CO₂H),4.24(t,J＝4.7Hz,2H,CH₂-O),3.56(t,J＝4.8Hz,2H,CH₂-O),3.25(s,3H,CH₃-O).¹³C NMR(101MHz,DMSO-d₆)δ(ppm):167.58(C＝O),160.35(C₂),152.71(C₆),149.59(C₄),148.29(C₈) 142.48,130.41,130.07,127.90 (benzene), 98.86 (C)₅),70.67(O-CH₂-),65.79(-CH₂-O),58.52(-OCH₃),42.64(CH₂-C₆H₄COOH) HRMS calcd C₁₆H₁₉N₅O₅ ⁺[M+H]⁺360.1302, found 360.1304.

Example 2: preparation of TLR7 agonist coupled with carrier protein BSA

Synthesis of compound 14: compound 5(0.03mmol) and EDCI (0.09mmol) were dissolved in DMF (1mL) under argon and NHS (N-hydroxysuccinimide) (0.09mmol) was added and stirred at 25 ℃ for 3 h; the resulting mixture was then spun dry with an oil pump to give compound 13.

BSA (0.3. mu. mol, available from Zhengmao scientific and engineering Co., Ltd., Wuhan city, under the trademark CC1050003) was dissolved in PBS (2mL), compound 13(0.006mmol) was dissolved in DMF, and the two solutions were mixed and reacted at 25 ℃ for 48 hours on a shaker. The resulting compound 14 (defined as TLR7a-BSA) was purified by centrifugation using an ultrafiltration tube (Millipore UFC 91009615M, 10KD) and lyophilized. The average number of covalent attachments of TLR7a to BSA was analytically calculated to be 6 to 7 using MALDI-TOF-MS testing.

Example 3: preparation of antigen MUC1

The compound 8 adopts manual solid-phase synthesis and comprises the following operation steps:

(1) activation of Rink Amide AM (available from Gill Biochemical (Shanghai) Co., Ltd.) resin

Rink Amide AM resin 306mg (0.2mmol) was weighed into a solid phase synthesis reaction tube and swollen with dry Dichloromethane (DCM) (4mL) under nitrogen for 30 min. Washing: using dry DCM (3X 3mL) and dry: (

Spherical molecular sieve dry) was washed alternately with N, N-Dimethylformamide (DMF) (3 × 3 mL).

(2) Deprotection of the Fmoc group

20% piperidine/DMF solution (3mL, 3X 5min) was added to the reaction tube with nitrogen agitation, washing: the resin was washed with DCM (3X 3mL) and DMF (3X 3mL) in alternating cycles.

(3) Kaiser-test

The Kaiser-test judges whether the amino group is deprotected by observing the color development of the resin. The developer components are mainly ninhydrin, phenol and pyridine.

Dipping a plurality of resin particles into a clean test tube by using a clean medicine spoon, sequentially adding two drops of color development reagent ninhydrin, phenol and pyridine, heating for 1min by using a 120 ℃ hot air gun, and observing the color of the resin particles. If the color is changed into blue, the amino group is exposed, and the Fmoc protecting group is removed; if there is no significant color change, it is indicated that the Fmoc protecting group is not removed, i.e., the amino group is not exposed.

(4) Coupling of amino acids

In a sample vial (10mL), the amino acid Fmoc-AA-OH (3.0 equiv.) and PyBOP (3.0 equiv.) were weighed and dried DMF solvent (2-3mL) was added to dissolve the amino acid completely. Then DIPEA (6.0 equivalent) is added and mixed evenly, and then the mixture is added into the resin which is cleaned and well swelled, and the reaction is carried out for about 2 hours at room temperature under the protection of nitrogen.

(5) Kaiser-test: adding color developing agent, heating with hot air gun, observing without obvious color change, indicating reaction is complete.

(6) Repeating the steps (1), (2), (3), (4) and (5) until the amino acid connection is completed.

(7) Removal of acetyl groups

Under nitrogen agitation, hydrazine hydrate was added: DMF: methanol 1:1:1(v/v/v,3mL, 2 × 15min), wash: the resin was washed with DCM (3X 3mL) and DMF (3X 3mL) in alternating cycles.

(8) The peptide chain is cleaved from the resin.

After peptide chain synthesis was complete, TFA/TIPS/H was used₂O (95:2.5:2.5, v/v/v) was deprotected completely for two hours, oil-pumped to dryness, and ether precipitated to give crude Compound 8 as a white crude solid.

(9) Analyzing, identifying and purifying the crude product.

Performing polypeptide solid phase synthesis by 306mg Rinke Amide AM resin, and directly using TFA/TIPS/H after the peptide chain is completely synthesized₂O (95:2.5:2.5, v/v/v) is completely deprotected, reacted for 2h, oil-pumped to dryness, and ether precipitated to obtain compound 8. Identified by HPLC, HRMS (EI) and NMR. HPLC chromatographic conditions: 5-90% of D liquid (CH)₃CN) in E solution (H)₂O + 0.1% TFA) at λ 220nm for 6.699min over 30 min. HRMS (EI data, Calculation C) for Compound 8₅₉H₉₈N₁₈O₂₃Calculated value of [ M + H + Na]²⁺The calculated value m/z is 725.3509, found value is 725.3215.¹H NMR(600MHz,D₂O)δ(ppm):4.87(d,1H,H₁),4.64–4.63(m,2H,D_α,R_α),4.59–4.57(m,2H,A_α),4.54–4.43(m,6H,P_α,S_α,T_α),4.29–4.27(m,1H,V_α),4.11–4.09(m,4H,T_β),4.05–4.02(m,1H,H₃),3.99–3.88(m,5H,G_α,H₅),3.86–3.63(m,12H,S_β,P_δ,H₂,H₄,H₆),3.50–3.23(m,2H,R_δ),2.96–2.79(m,2H,D_β),2.34–2.29(m,3H,P_β1),2.16–2.08(m,1H,V_β),2.05–2.01(m,6H,P_β2,Ac-NH),2.00–1.87(m,6H,P_γ),1.73–1.71(m,4H,R_β,R_γ),1.42–1.33(m,6H,A_β),1.27–1.23(m,6H,T_γ),0.99–0.97(m,6H,V_γ).¹³C NMR(151MHz,D₂O)δ(ppm):174.79,174.08,173.73,173.70,173.48,173.45,173.16,173.05,172.65,171.47,171.07,170.95,170.74,167.22,163.05(15×C＝O),156.65(R_ζ),98.52(C₁),75.36(C₅),71.32(C₄),68.45(C₃),67.96,67.94(T_β),66.96(C₆),61.26(S_β),60.97,60.65,60.27(P_α),59.88(V_α),59.56,58.87(T_α),57.04(S_α),55.15(C₂),51.13(R_α),50.22(D_α),49.59,47.83,47.74(P_δ),47.68,47.58(A_α),41.99(R_δ),40.40,40.26(G_α),36.30(D_β),30.09(V_β),29.34(R_β),29.22,29.17,27.28(P_β),24.64(R_γ),24.56,24.50,24.08(P_γ),22.25(Ac-NH),18.68,18.32(T_γ),17.37(V_γ),15.43,15.09(A_β)。

Example 4: preparation of antigen MUC1 squaric acid monoamide

The compound 10 is synthesized by adopting an artificial solid phase, and the operation steps are as follows:

(1) deprotection of Fmoc group of resin 7

(2) Kaiser-test

(3) Coupling of amino acids

In a sample vial (10mL), the amino acid Fmoc-Gly-OH (3.0 equiv.) and PyBOP (3.0 equiv.) were weighed and dried DMF solvent (2-3mL) was added to dissolve the amino acid completely. Then DIPEA (6.0 equivalent) is added and mixed evenly, and then the mixture is added into the resin which is cleaned and well swelled, and the reaction is carried out for about 2 hours at room temperature under the protection of nitrogen.

(4) Kaiser-test: adding color developing agent, heating with hot air gun, observing without obvious color change, indicating reaction is complete.

(5) And (3) repeating the steps (1) and (2).

(6) Removal of acetyl groups

(7) Diethyl squarate (6.0 equiv) and DIPEA (6.0 equiv) are added into dry DMF solvent (2-3mL) and mixed evenly to react for about 2h at room temperature under the protection of nitrogen.

(8) The peptide chain is cleaved from the resin.

After peptide chain synthesis was complete, TFA/TIPS/H was used₂O (95:2.5:2.5, v/v/v) was deprotected completely for two hours, oil-pumped to dryness, and ether precipitated to give crude Compound 10 as a white crude solid.

(9) Analyzing, identifying and purifying the crude product.

Peptide solid phase synthesis was performed by 306mg of Rinke Amide AM resin, and after peptide chain synthesis was complete, TFA/TIPS/H was used directly₂O (95:2.5:2.5, v/v/v) is completely deprotected, reacted for two hours, oil-pumped to dryness, and ether precipitated to give compound 10. Identified by HPLC, HRMS and NMR. HPLC chromatographic conditions: 5-90% of D liquid (CH)₃CN) in E solution (H)₂O + 0.1% TFA) at λ 220nm for 7.152min over 30 min. HRMS (EI) data for Compound 10, calcd for C₆₇H₁₀₅N₁₉O₂₇，[M+2H]²⁺The calculated value m/z is 805.3803, found 805.3833. [ M + H + Na ]]²⁺The calculated value m/z is 816.3713, found 816.3729.¹H NMR(600MHz,CD₄O)δ(ppm):4.81(d,1H,H₁),4.77–4.71(m,2H,D_α,R_α),4.63–4.56(m,2H,A_α),4.48–4.45(m,2H,-CH₂-),4.43–4.34(m,6H,P_α,S_α,T_α),4.23–4.21(m,2H,V_α),4.17(m,1H,T_β),4.12–4.08(m,1H,H₃),3.99–3.83(m,7H,G_α,H₅),3.81–3.60(m,12H,S_β,P_δ,H₂,H₄,H₆),3.26–3.17(m,2H,R_δ),2.95–2.83(m,3H,D_β),2.27–2.20(m,3H,P_β1),2.13–2.11(m,1H,V_β),2.07–1.99(m,6H,P_β2,Ac-NH),1.99–1.86(m,6H,P_γ),1.70–1.67(m,4H,R_β,R_γ),1.47–1.43(m,3H,CH₃-),1.42–1.33(m,6H,A_β),1.26–1.18(m,6H,T_γ),1.00–0.98(m,6H,V_γ).¹³C NMR(151MHz,CD₄O)δ(ppm):188.92,183.79,177.49,174.25,173.78,173.56,173.10,173.04,173.00,172.65,172.60,172.23,172.09,172.05,171.96,170.84,170.67,170.62,170.30(18C＝O),160.71,160.46(C＝C),157.11(R_ζ),98.56(C₁),74.25(C₅),71.62(C₄),69.56(C₃),69.50,68.86(T_β),68.70(C₆),66.69(-CH₂-),61.48(S_β),61.35,60.72,59.83(P_α),56.91(V_α),56.33,56.19(T_α),56.05(S_α),55.91(C₂),55.76(R_α),55.31(D_α),50.57,50.57,50.04(P_δ),49.83,48.15(A_α),46.03(R_δ),42.00,41.84,40.62(G_α),34.71(D_β),30.00(V_β),29.11(R_β),29.03,28.96,27.99(P_β),24.76(R_γ),24.70,24.52,24.42(P_γ),22.05(Ac-NH),18.36,18.17(T_γ),15.99,15.86(V_γ),15.74,15.30(A_β)。

Example 5: preparation of antigen MUC1 squaric acid monoamide coupled with carrier protein BSA

Synthesis of compound 12: compound 10(0.03mmol) and BSA (0.06 μmol) were dissolved in 5mL of 0.07M Na at pH 9.5₂B₄O₇/0.035M KHCO₃In a buffer. After 48h reaction at 25 ℃ on a shaker, the resulting compound 12 (defined as BSA-MUC1) was purified by centrifugation through an ultrafiltration tube (Millipore UFC 91009615M, 10KD) and lyophilized. The average number of covalent attachments of MUC1 to BSA was analytically calculated to be 9 to 11 by MALDI-TOF-MS testing.

Example 6: the preparation of the BSA-MUC1 conjugate and the TLR7 agonist conjugate defines that the anti-tumor vaccine molecule is TLR7a-BSA-MUC 1.

Synthesis of compound 15: BSA-MUC1 (Compound 12) (0.12. mu. mol) was dissolved in PBS (1mL), Compound 13(0.006mmol) dissolved in DMF was added and reacted at 25 ℃ for 48h on a shaker to give Compound 15(TLR7a-BSA-MUC1) which was purified by centrifugation through an ultrafiltration tube (Millipore UFC 91009615M, 10KD) and lyophilized. The average number of covalent linkages of TLR7a to BSA-MUC1 was analytically calculated to be 6 to 7 using MALDI-TOF-MS assays.

Example 7: preparation of coating antigen biotin-MUC 1

The compound 11 is synthesized by manual solid phase synthesis, and the operation steps are as follows:

(1) deprotection of Fmoc group of resin 9

(2) Kaiser-test

Dipping a plurality of resin particles into a clean test tube by using a clean medicine spoon, sequentially adding two drops of color development reagent ninhydrin, phenol and pyridine, heating for 2min by using a 150 ℃ hot air gun, and observing the color of the resin particles. If the color is changed into blue, the amino group is exposed, and the Fmoc protecting group is removed; if there is no significant color change, it is indicated that the Fmoc protecting group is not removed, i.e., the amino group is not exposed.

(3) Coupling of biotin

In a sample vial (10mL), the amino acids biotin (3.0 equiv.), HATU (2.0 equiv.) and HOAt (2.0 equiv.) were weighed into dry DMF solvent (2-3mL) to completely dissolve the amino acids. Then DIPEA (6.0 equivalent) is added and mixed evenly, and then the mixture is added into the resin which is cleaned and well swelled, and the reaction is carried out for about 2 hours at room temperature under the protection of nitrogen.

(5) Removal of acetyl groups

(6) The peptide chain is cleaved from the resin.

After peptide chain synthesis was complete, TFA/TIPS/H was used₂O (95:2.5:2.5, v/v/v) was deprotected completely for two hours, oil-pumped to dryness, and ether precipitated to give crude Compound 11 as a white crude solid.

(7) Analyzing, identifying and purifying the crude product.

Peptide solid phase synthesis was performed by 306mg of Rinke Amide AM resin, and after peptide chain synthesis was complete, TFA/TIPS/H was used directly₂O (95:2.5:2.5, v/v/v) is completely deprotected, reacted for 2h, oil-pumped to dryness, and ether precipitated to obtain compound 11. HPLC, HRMS and NMR identification, HPLC chromatographic conditions: 5-60% of D liquid (CH)₃CN) in E solution (H)₂O + 0.1% TFA) at 220nm for 20min, a retention time of 8.498 min. HRMS (EI) data for Biotin-MUC 1 glycopeptide 11: calculated value C₇₁H₁₁₅N₂₁O₂₆S，[M+H+Na]²⁺Calculated value M/z 866.9004 found 866.8993.[ M +2Na]²⁺The calculated value m/z is 877.8914, found 877.8909.¹H NMR(600MHz,CD₄O)δ(ppm):4.80(d,1H,H₁),4.77–4.74(m,2H,H₁₃,H₁₄),4.62–4.55(m,2H,D_α,R_α),4.51–4.41(m,6H,P_α,S_α,T_α),4.37–4.25(m,5H,V_α,T_β),4.21–4.19(m,1H,H₃),4.00–3.87(m,7H,G_α,H₅),3.87–3.65(m,12H,S_β,P_δ,H₂,H₄,H₆),3.65–3.60(m,1H,H₁₃),3.24–3.18(m,2H,R_δ),2.95–2.91(m,2H,D_β),2.87–2.69(m,2H,H₁₂),2.32–2.29(m,2H,H₇),2.26–2.18(m,3H,P_β1),2.12–2.10(m,V_β),2.05–1.87(m,12H,P_β2,Ac-NH,P_γ),1.87–1.84(m,1H,H_10’),1.75–1.73(m,2H,H₈),1.70–1.67(m,4H,R_β,R_γ),1.48–1.46(m,2H,H₉),1.33–1.30(m,1H,H_10’),1.25–1.20(m,6H,T_γ),1.01–0.98(m,6H,V_γ).¹³C NMR(151MHz,CD₄O)δ(ppm):176.71,174.78,174.32,174.27,174.22,173.94,173.87,173.82,173.48,173.32,173.19,172.47,172.13,172.11,171.92,171.86,171.54,165.97(18C＝O),158.34(R_ζ),99.80(C₁),75.48(C₅),72.84(C₄),70.08(C₃),69.91,67.88(T_β),63.04(C₆),62.56(C₁₃),61.94(S_β),61.49,61.19,61.05(P_α),60.23(C₁₄),58.14(V_α),57.41,57.28(T_α),57.13(S_α),56.99(C₁₁),56.76(C₂),56.55(R_α),54.40(D_α),51.80,51.27,51.05(P_δ),49.77,49.37(A_α),43.61(R_δ),43.46,43.06,41.85(G_α),40.85(C₁₂),36.20(C₇),35.95(D_β),31.17(V_β),30.34,30.26,30.19(P_β),29.43(C₉),29.23(C₁₀),26.35(C₈),25.99(R_γ),25.94,25.75,25.65(P_γ),23.28(Ac-NH),19.88,19.59(T_γ),18.73(V_γ),16.58,16.54(A_β)。

Example 8: immunization of mice

35 female BALB/c mice 6 to 8 weeks old were purchased (from the animal laboratories, university of agriculture, Wash.). The specific immunization time course is respectively day 1, day 15 and day 29, and the injection mode is intraperitoneal injection. The blood sampling mode is tail breaking blood sampling, and the blood sampling time is as follows: blank blood was taken before immunization, 2h after the first immunization, and 14 days after each immunization injection, with the last (day 42) being orbital bleeding. After blood was removed, serum was removed by centrifugation in a centrifuge and stored at-80 ℃.

Table 1: composition of each vaccine group

Example 9: in vivo cytokine assay

(1) Antigen coating: 100. mu.L of diluted capture antibody (200-fold diluted with coating solution) was added to each well, and after the addition was complete, the 96-well plate was sealed with a preservative film and incubated overnight in a refrigerator at 4 ℃.

(2) Washing: the 96-well plate was taken out of the refrigerator and returned to room temperature (25 ℃ C., the same applies hereinafter), the coating liquid was thrown off, and the residual liquid was gently blotted with absorbent paper. Add 200. mu.L of PBST solution to each well, spin off the wash solution, and blot dry with absorbent paper. Repeat 4 times.

(3) Filling protein: 200 μ L of 1% BSA in PBS (w/v) was added to each well, and after the addition was complete, it was sealed with a preservative film and shaken at room temperature for 1h, and washed as above.

(4) Add standard/serum:

a) standards diluted by concentration gradient were added according to kit instructions.

b) Serum was diluted 20-fold with diluent, 100. mu.L per well, and two duplicate wells were set. The mixture is sealed by a preservative film and placed at room temperature for 2 hours. The washing was as above.

(5) Adding a detection antibody: 100 μ L of diluted detection antibody (200-fold diluted with diluent) was added to each well. After the liquid adding is finished, the mixture is sealed by a preservative film and placed at room temperature to shake for 1 h. And (6) washing.

(6) Add Avidin-HRP: mu.L of diluted Avidin-HRP (1000-fold dilution with diluent) was added to each well. After the liquid is added, the mixture is sealed by a preservative film and placed at room temperature for 30 min.

(7) After washing, 200. mu.L of PBST solution was added to each well and allowed to stand for 30min, the washing solution was thrown off and blotted dry with absorbent paper. Repeat 5 times.

(8) Substrate color development reaction: after the color developing solution A and the color developing solution B are mixed uniformly in equal volume, 100 mu L of each hole is added into a 96-hole plate. The 96-well plate was shaken at room temperature in the dark for 30 min. Finally, 100. mu.L of stop solution is added to each well. The 96-well plate was immediately placed in a microplate reader, and the absorbance at 450nm was measured. And drawing a standard curve by using the absorbance value of the standard substance, and substituting the absorbance value of the serum into the standard curve to obtain the content of the cell factor in the serum.

FIG. 2 shows sera taken 2h after the first immunization for IFN-. gamma.and IL-6 assays, respectively, each bar representing the mean of the content of 5 mice, sera from each mouse being independently repeated three times, the mean being taken for the three values, and the mean Standard Error (SEM) being indicated by the error bars. Significant differences were compared to the PBS group: p < 0.01; p < 0.0001; ns, significant differences were not significant. The vaccines were compared with each other: p < 0.01; p < 0.0001; ns, significant differences were not significant.

Example 10: indirect non-competitive ELISA method for determining antibody content in blood

(1) Coating: the coating antigen biotin-MUC 1 was mixed with avidin (avidin) at a molar ratio of 4:1, diluted with a coating solution at pH 9.5 and an antigen (MUC1) concentration of 0.1 μ g/mL, 100 μ L per well, and placed in a refrigerator at 4 ℃ overnight.

(2) Washing: the 96-well plate was taken out of the refrigerator to return to room temperature, the coating solution was thrown out, and the residual liquid was blotted with absorbent paper. Add 200. mu.L of PBST solution to each well, spin off the wash solution, and blot dry with absorbent paper. Repeat 3 times.

(3) And (3) sealing: mu.L of 1% casein PBS buffer (the concentration of PBS buffer is in mass percent, the same applies below) was added to each well, incubated in an incubator at 37 ℃ for 1 hour, and washed as above.

(4) Antigen-antibody specific binding: the serum was diluted with 0.1% casein in PBS buffer at 200, 400, 800, 1600, 3200, 64000, 128000, 256000, 512000, 1024000, 2048000, 4096000, 8192000, 16384000. 100 μ L of diluent was added to each well, and blanks and negative controls were set. Incubate at 37 ℃ for 1 h. The washing was as above.

(5) Adding a secondary antibody: the goat anti-mouse IgG-HRP was dissolved in PBST buffer, diluted to 5000-fold with PBST buffer, mixed well, 100. mu.L of the dilution was added to each well, and incubated at 37 ℃ for 1h in an incubator. Secondary antibodies (IgM-HRP, IgG2a-HRP, IgG2b-HRP, IgG1-HRP, IgG3-HRP, IgA-HRP, IgE-HRP) were added in the same manner. The washing was as above.

(6) Color development: add 100. mu.L of color reagent into each well (one plate needs 9.5mL +0.5mLF solution (2mg/mL TMB/absolute ethyl alcohol) +32uLG solution (35% urea hydrogen peroxide/aqueous solution)), shake and mix, store for 5min at room temperature in dark place.

(7) And (4) terminating: add 50. mu.L of stop solution (2M sulfuric acid) to each well, mix well in a microplate reader, read, and measure the absorbance of each well at a wavelength of 450 nm.

Figure 3 is an assessment of the titer of anti-MUC 1 produced by the vaccine (IgG antibody titer test) for sera obtained after the 3 rd immunization. Each bar represents the mean of the titers of 5 mice, and each value is independently repeated three times, with the mean standard error being identified by the error bars. Significant differences were compared to the PBS group: p < 0.01; p < 0.001; ns, significant differences were not significant. The vaccines were compared with each other: p < 0.01; p < 0.001; ns, significant differences were not significant.

Figure 4 shows the evaluation of the IgG titers produced by the vaccine against MUC1 for sera obtained after 1 st, 2 nd, and 3 rd immunizations. Each bar represents the mean of the titers of 5 mice, and each value is independently repeated three times, with the mean standard error being identified by the error bars.

Figure 5 shows the IgM titers generated by the vaccine against MUC1 for sera obtained after 1 st, 2 nd, 3 rd immunizations. Each bar represents the mean of the titers of 5 mice, and each value is independently repeated three times, with the mean standard error being identified by the error bars.

Figure 6 shows the evaluation of the anti-MUC 1 antibody subtype produced by the vaccine for sera obtained after the 3 rd immunization. Each bar represents the mean of the titers of 5 mice, and each value is independently repeated three times, with the mean standard error being identified by the error bars. Significant differences were compared to the PBS group: ns, no significant difference; p < 0.0001.

Example 11: indirect non-competitive ELISA method for determining antibody content in blood

(1) Coating: antigen BSA was coated and diluted with pH 9.5 coating solution at 1.16 μ g/mL antigen (BSA) concentration, 100 μ L per well, and placed in a refrigerator at 4 ℃ overnight.

(3) And (3) sealing: mu.L of 1% casein in PBS buffer was added to each well, incubated at 37 ℃ for 1h in an incubator, and washed as above.

(5) Adding a secondary antibody: the goat anti-mouse IgG-HRP was dissolved in PBST buffer, diluted 4000-fold with PBST buffer, mixed well, 100. mu.L of the dilution was added to each well, and incubated at 37 ℃ for 1h in an incubator. The washing was as above.

(6) Color development: add 100. mu.L of color developing solution (one plate requires 9.5mL +0.5mL of F solution (2mg/mL TMB/absolute ethanol) +32uL of G solution (35% urea hydrogen peroxide/aqueous solution)) to each well, shake and mix well, and store in dark at room temperature for 5 min.

Figure 7 shows the evaluation of anti-BSA titers generated by vaccines using BSA coated plates for sera obtained after the 3 rd immunization. Each bar represents the mean of the titers of 5 mice, and each value is independently repeated three times, with the mean standard error being identified by the error bars. Significant differences were compared to the PBS group: p < 0.05; p < 0.01; p < 0.0001. The vaccines were compared with each other: p < 0.05; p < 0.0001; ns, significant differences were not significant.

Example 12: MTT method for determining MCF-7 cell viability

This example aims to investigate whether antibodies are able to mediate complement lysis by activating CDC.

(1) MCF-7 cells were trypsinized, homogenized by adding 10% by volume FBS/DMEM, and transferred to 96-well cell culture plates (8000 cells per well).

(2) Then washed 3 times with PBS and 1% BSA/PBS (50. mu.L/well) was added to the diluted mouse serum at 1: 50. The cells were kept in the incubator for 2 h.

(3) Rabbit complement (1:50 dilution) 1% BSA/PBS (RC for rabbit complement; HIRC for heat inactivated rabbit complement) was then added (50. mu.L/well) and incubated for 4 h.

(4) 0.5% MTT/PBS solution was prepared for addition (20. mu.L/well) and incubated at 37 ℃ for 2 h.

(5) Finally, the supernatant was aspirated, DMSO was added (150. mu.L/well), and the mixture was repeatedly blown and beaten several times to mix well, and the absorbance was measured at 490 nm. The cell viability of MCF-7 cells was determined by the following equation:

cell viability (%). gtoreq (experiment/control). times.100%

FIG. 8 is a graph of the MTT assay for assessing MCF-7 cell viability for sera obtained after the 3 rd immunization. Each bar represents the mean of 5 mice, and each value is independently repeated three times, with the error bars identifying the mean standard error. Significant differences were compared to the PBS group: p < 0.05; p <0.001, P <0.0001, ns, no significant difference was evident. The vaccines were compared with each other: p < 0.05; p < 0.0001.

Example 13: flow cytometry (FACS) determination of antibody binding to MCF-7 cells

Detection of candidate vaccine-induced antibody binding to MCF-7 cells, staining of cells with fluorescent secondary antibody, followed by flow cytometry (FACS) analysis.

(1) MCF-7 cells were trypsinized and then transferred to different conical centrifuge tubes (1X 10) by adding medium⁶Cells/tube), centrifuge 1500g for 2min, aspirate supernatant.

(2) Wash (1% BSA/PBS) (300. mu.L/tube) was added, centrifuged, the cells were settled at the bottom of the tube, the supernatant aspirated, and repeated 3 times. Mouse serum samples (pooled blood of 5 mice) were diluted 50-fold (250 μ L/tube) with FACS solution and incubated at 0 ℃ for 60 min.

(3) The supernatant was centrifuged off. Wash solution (300. mu.L/tube) was added, centrifuged, the cells were settled at the bottom of the tube, the supernatant aspirated, and repeated 3 times. The fluorescently labeled secondary antibody was diluted 50-fold with FACS solution (100. mu.L/tube) and incubated at 0 ℃ for 30 min.

(4) The supernatant was centrifuged off. Wash solution (300. mu.L/tube) was added, centrifuged, the cells were settled at the bottom of the tube, the supernatant aspirated, and repeated 3 times. Finally, each tube was dissolved in 250. mu.L of FACS solution and mixed well.

(5) The fluorescence intensity was measured by flow cytometry.

FIG. 9 shows flow cytometry (FACS) analysis of immune mouse antisera binding to MCF-7 cancer cells. PBS group (black) was used as control. These images are representative of five independent experiments.

Example 14: confocal microscopy determination of antibody binding to MCF-7 cells

MCF-7 cells were stained with mouse serum to determine their potential to recognize the MUC1 target.

(1) First, MCF-7 cells were trypsinized, added to the medium, and then transferred to a confocal dish (1X 10)⁶Individual cells/dish) into a cell incubator for 12 h.

(2) The medium was aspirated, washed 3 times with 1% BSA/PBS solution, and then the mouse serum was diluted 50-fold (500. mu.L/dish) with 1% BSA/PBS solution and incubated for 1h at 0 ℃.

(3) The supernatant was aspirated, washed 3 times with 1% BSA/PBS, and the fluorescently labeled secondary antibody was diluted 50-fold (500. mu.L/dish) with 1% BSA/PBS and incubated at 0 ℃ for 30 min.

(4) The cells were washed 3 times with 1% BSA/PBS, then 2 times with PBS, and finally 500. mu.L PBS was added and the MCF-7 cells were observed under a confocal microscope (Leica TCS SP8, Wetzlar, Germany) with 63 fold oil lens and photographed.

Example 15: CTL assay

(1) Each group of BALB/c mice (n ═ 5) were immunized 3 times with subcutaneous injections of the vaccine candidates on days 1, 15 and 29, respectively. 14 days after the third immunization, the spleen of the mouse is taken to prepare a single cell suspension which is used as an effector cell for CTL detection.

(2) Freshly isolated splenocytes (1X 10)⁶One cell/well) was added to RPMI-1640, and MCF-7 cells (1X 10)⁶Individual cells/well) were incubated for 12 h. The cytotoxicity of effector cells against target cells was then detected using Lactate Dehydrogenase (LDH).

(3) And (3) taking out the cell culture plate from the cell culture box 1h before the preset detection time point, adding an LDH releasing agent (10% of the original culture solution volume) into the sample maximum enzyme activity control hole, repeatedly blowing and beating the sample maximum enzyme activity control hole for a plurality of times after adding the LDH releasing agent, uniformly mixing the mixture, and then continuously incubating the mixture in the culture box for 1 h.

(4) After the predetermined time is reached, the cell culture plate is centrifuged for 4min in a multi-well centrifuge 250g centrifuge. Then, 120. mu.L of the supernatant from each well was pipetted onto another 96-well microplate, and then LDH detection working solution (60. mu.L/well) was added. The 96-well plate was incubated at room temperature for 30min in the absence of light, and the absorbance was measured at 490 nm. Meanwhile, MCF-7 cells were incubated alone and splenocytes were incubated alone to determine spontaneous LDH release values. MCF-7 cells were incubated in RPMI-1640 without FBS lysate and the maximum release of LDH was determined. The cell lysis rate was calculated as follows:

cytotoxicity (%) - (experimental group-absorbance of target cell spontaneous/maximum enzyme activity in cell-target cell spontaneous) × 100%

FIG. 10 shows CTL assays evaluating splenocytes cytotoxicity against MCF-7 cells in vitro after 3 rd immunization. Each bar represents the mean of 5 mice, and the error bars identify the mean standard error. The vaccines were compared with each other: p < 0.05; p < 0.01.

From the vaccine immunity test results, the anti-tumor vaccine molecules provided by the invention generate IgG antibodies with high affinity and aiming at tumor-associated antigens or specific antigens. The antibodies produced recognize cancer cells and initiate lysis of the recognized cancer cells by activating Complement Dependent Cytotoxicity (CDC) of rabbit serum. These results indicate that the design of the strategy of the in-line adjuvant protein conjugate provided by the present invention is an effective strategy for designing a highly effective immunotherapeutic anti-cancer vaccine.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. An anti-tumor vaccine molecule with a structure shown in a formula (I), wherein in the formula (I), A is an adjuvant, B is an antigen, m A are respectively and covalently connected with the protein through at least one covalent linking arm, n B are respectively and covalently connected with the protein through at least one covalent linking arm, and the number of amino acid molecules in the protein is more than or equal to 100; m in the formula (I) is an integer which is more than or equal to 1, and n is an integer which is more than or equal to 1;

A_m-protein-B_nFormula (I);

preferably, m in the formula (I) is 1, n is an integer of 2 or more;

preferably, m in the formula (I) is an integer of 2 or more, and n is an integer of 2 or more.

2. The anti-tumor vaccine molecule according to claim 1, wherein the antigen comprises at least one of a tumor-associated antigen, a tumor-specific antigen, a pathogen antigen, a biotoxin, and a biomolecule antigen;

3. The anti-tumor vaccine molecule according to claim 2, wherein the tumor-associated polypeptide antigen or the tumor-associated glycopeptide antigen comprises at least one selected from the group consisting of MUC1, MUC16, NY-ESO-1, MAGE-a1/3/4, WT1, STAT3, HER2 and GP 100.

4. The anti-tumor vaccine molecule according to claim 2, wherein the antigen comprises MUC1, preferably the MUC1 antigen is selected from at least one of the following structures:

wherein, in the structure of the antigen containing MUC1, each R¹、R²、R³、R⁴And R⁵Each independently selected from hydrogen and the sugar structures shown below:

5. the anti-tumor vaccine molecule according to claim 2, wherein the tumor-associated carbohydrate antigen is selected from at least one of the following structures:

6. the anti-tumor vaccine molecule according to any one of claims 1 to 5, wherein the protein is selected from at least one of bovine serum albumin, chicken egg albumin, keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, Haemophilus influenzae D protein, group B meningococcal outer membrane protein complex, pertussis toxoid, typhoid bacillus flagellae, pneumolysin, and a non-toxic diphtheria toxin mutant.

7. The anti-tumor vaccine molecule according to any one of claims 1-6, wherein the adjuvant is a pattern recognition receptor agonist;

preferably, the pattern recognition receptor agonist is selected from at least one of a Toll-like receptor agonist and an NKT agonist;

preferably, the Toll-like receptor agonist is selected from at least one of a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR3 agonist, a TLR2 agonist and a TLR4 agonist.

8. The anti-tumor vaccine molecule according to any one of claims 1 to 5, wherein the structure of each of the covalently linked arms is independently selected from the following structures:

-CO-、-O-CO-、-NH-CO-、-NH(C＝NH)-、-SO₂-、-O-SO₂-、-NH-、-NH-CO-CH₂-、-CH₂-、-C₂H₄-、-C₃H₆-、-C₄H₈-、-C₅H₁₀-、-C₆H₁₂-、-C₇H₁₄-、-C₈H₁₆-、-C₉H₁₈-、-C₁₀H₂₀-、-CH(CH₃)-、-C[(CH₃)₂]-、-CH₂-CH(CH₃)-、-CH(CH₃)-CH₂-、-CH(CH₃)-C₂H₄-、-CH₂-CH(CH₃)-CH₂-、-C₂H₄-CH(CH₃)-、-CH₂-C[(CH₃)₂]-、-C[(CH₃)₂]-CH₂-、-CH(CH₃)-CH(CH₃)-、-C[(C₂H₅)(CH₃)]-、-CH(C₃H₇)-、-(CH₂-CH₂-O)_p-CH₂-CH₂-、-CO-CH₂-、-CO-C₂H₄-、-CO-C₃H₆-、-CO-C₄H₈-、-CO-C₅H₁₀-、-CO-C₆H₁₂-、-CO-C₇H₁₄-、-CO-C₈H₁₆-、-CO-C₉H₁₈-、-CO-C₁₀H₂₀-、-CO-CH(CH₃)-、-CO-C[(CH₃)₂]-、-CO-CH₂-CH(CH₃)-、-CO-CH(CH₃)-CH₂-、-CO-CH(CH₃)-C₂H₄-、-CO-CH₂-CH(CH₃)-CH₂-、-CO-C₂H₄-CH(CH₃)-、-CO-CH₂-C[(CH₃)₂]-、-CO-C[(CH₃)₂]-CH₂-、-CO-CH(CH₃)-CH(CH₃)-、-CO-C[(C₂H₅)(CH₃)]-、-CO-CH(C₃H₇) -and-CO- (CH)₂-CH₂-O)_p-CH₂-CH₂-；

Wherein, in the structure of the covalent linking arm,

each x is independently selected from an integer from 1 to 60;

each Y is independently selected from at least one of-NH-, -O-, -S-and-S-S-;

each p is independently selected from an integer from 1 to 60.

9. A method of preparing an anti-tumor vaccine molecule according to any one of claims 1 to 8, comprising:

10. Use of an anti-tumor vaccine molecule according to any one of claims 1 to 8 in an anti-tumor vaccine.